Functional Consequences of Sequence Variation in Bundlin, the Enteropathogenic Escherichia coli Type IV Pilin Protein

Abstract

The bundle-forming pilus (BFP) of enteropathogenic Escherichia coli (EPEC) is an important virulence factor. We examined the role of divergent alleles of bfpA encoding bundlin, the BFP pilin protein, in pilus biogenesis, pilus interactions, and immune responses. We found that the BFP biogenesis machine from an EPEC strain that expresses one bundlin type is capable of assembling all other bundlin types. Furthermore, we found that EPEC strains expressing divergent bundlin types are capable of forming mixed autoaggregates, suggesting that different pilin types can intertwine. However, we found that there was a marked difference between alleles in immunogenicity in both rabbits and mice of a peptide derived from a region of bundlin undergoing apparent diversifying selection. In addition, despite a high degree of cross-reactivity between divergent bundlin proteins, in both mice and rabbits responses appeared to be stronger against the homologous pilin protein than against the heterologous protein. This result was verified using sera from a volunteer study, which demonstrated that the human antibody responses after an initial challenge with live EPEC were stronger against the homologous bundlin protein than against a divergent bundlin protein. However, a repeat challenge induced equivalent responses. These results are consistent with the hypothesis that human immune responses against bundlin exert selective pressure on bfpA sequence divergence.